Differential Interference Contrast Microscopy of Human Atherosclerotic Lesions

Differential interference contrast microscopy was employed to study sections of human cerebral arteries and aorta. When this procedure was used to observe 0.5-μ-thick sections of plastic-embedded arteries, images were obtained with greater definition of detail than corresponding micrographs of the area using conventional bright-field microscopy. Since structures with different refractive indices are shown in geometric relief from one another, an apparent three-dimensional image is seen, which, together with the theoretically higher resolution and contrast achieved by this technic, gives an image with definition between conventional light and low magnification electron microscopy. The morphology of fatty streak lesions with aggregates of round and elongated lipid-filled cells was demonstrated, and clear images of different forms and sizes of intracellular lipid droplets were illustrated at magnifications around 1000. In atheromatous plaques, intra- and extracellular lipid droplets were observed, some with partially extracted cores or surfaces, as were amorphous lipid droplets coalescing with crystals whose centers had been extracted. We conclude that this optical accessory to a research photomicroscope is a valuable supplemental tool in studies on the morphology of atherosclerotic lesions.     

Augmented Expression of Cyclooxygenase-2 in Human Atherosclerotic Lesions

Cyclooxygenase-1 (Cox-1) and Cox-2 convert arachidonic acid to prostaglandin H(2), the precursor of other prostaglandins and thromboxanes, eicosanoids important in vascular pathophysiology. However, knowledge of the expression of cyclooxygenases within atherosclerotic lesions is scant. This study tested the hypothesis that human atheroma and nonatherosclerotic arteries express the two Cox isoforms differentially. Cox-1 mRNA and protein localized on endothelial and medial smooth muscle cells of normal arteries (n = 5), whereas Cox-2 expression was not detectable. In contrast, atheromatous (n = 7) lesions contained both Cox-1 and Cox-2, colocalizing mainly with macrophages of the shoulder region and lipid core periphery, whereas smooth muscle cells showed lower levels, as demonstrated by immunohistochemical and in situ hybridization analysis. Furthermore, microvascular endothelium in plaques showed notable staining for both isoforms. In accord with immunohistochemical studies, Western blot analysis of protein extracts from normal arteries revealed constitutive Cox-1, but not Cox-2, expression. Extracts of atheromatous lesions, however, contained both Cox-1 and Cox-2 protein, detected as two immunoreactive proteins of approximately 70 and 50 kd. Macrophages expressed the short form of Cox-1/-2 constitutively after several days of in vitro culture, rather than the 70-kd protein. These results shed new light on the inflammatory pathways that operate in human atheroma. In particular, the expression of Cox-2 in atheromatous, but not in unaffected, arteries has therapeutic implications, given the advent of selective Cox-2 inhibitors.     

Calcification Locates to Transglutaminases in Advanced Human Atherosclerotic Lesions

Transglutaminases play an important role in vascular smooth muscle cell-induced calcification in vitro. In this study, we determined whether these enzymes are also involved in human atherosclerotic calcification using nine carotid artery specimens obtained at endarterectomy. Sections of the carotid artery specimens were registered to micro-computed tomography images and stained for tissue-type transglutaminase, plasma transglutaminase factor XIIIA (FXIIIA), the N^ε(γ-glutamyl)lysine cross-link, and the macrophage marker CD68. Ex vivo micro-computed tomography revealed extensive calcification, which significantly correlated with the cross-link. FXIIIA was found to be the dominant transglutaminase, rather than tissue-type transglutaminase, although staining of both transglutaminases correlated with the cross-link. Staining for FXIIIA colocalized with CD68 at both the cellular and tissue level. In conclusion, areas of calcification locate to the presence and activity of transglutaminases in human atherosclerotic arteries. FXIIIA seems to be the dominant transglutaminase and may be derived from local macrophages. These results are consistent with the hypothesis that transglutaminases participate in the calcification process of human atherosclerotic arteries.Vascular calcification is considered a tightly regulated process of matrix deposition by osteoblast-like cells.¹ These cells may be derived from stem cells or differentiate from smooth muscle cells or pericytes. Recent work from Johnson et al² suggests a role for transglutaminases in the process of arterial calcification. On the basis of in vitro experiments on cultured healthy arterial segments and smooth muscle cells, these authors proposed a novel mechanism of arterial calcification, for which tissue-type transglutaminase (TG2) is central in the chondro-osseous differentiation and calcification of smooth muscle cells. Transglutaminases form a class of enzymes with pleiotropic function³ that consists of nine known members. Among these, TG2 is expressed in endothelial cells, smooth muscle cells, and monocytes/macrophages. The plasma transglutaminase, factor XIIIA (FXIIIA) is expressed in monocytes/macrophages.⁴ TG2 has been implicated in an animal model for atherosclerotic lesion formation,⁵ whereas both TG2 and FXIIIA are involved in vascular remodeling.^6,7 TG2 and FXIIIA are also involved in normal bone formation, where they act in concert to promote chondrocyte maturation.⁸ Although these data suggest an important role of transglutaminases in the development of atherosclerotic calcification, evidence of their role in human atherosclerotic lesion development is lacking. Therefore, we investigated the presence of transglutaminases, ie, TG2 and FXIIIA, the transglutaminase-induced cross-link and macrophages in human atherosclerotic carotid arteries. These markers were correlated to calcified areas that were detected ex vivo with micro-computed tomography (μCT) imaging.     

Characterization of Two Unique Cholesterol-Rich Lipid Particles Isolated from Human Atherosclerotic Lesions

The authors' laboratory, using histochemical methods, previously identified two types of cholesterol-containing lipid particles in the extracellular spaces of human atherosclerotic lesions, one particle enriched in esterified cholesterol and the other particle enriched in unesterified cholesterol. The authors isolated and characterized these lipid particles. The esterified cholesterol-rich lipid particle was a small lipid droplet and differed from intracellular lipid droplets found in foam cells with respect to size and chemical composition. It had an esterified cholesterol core surrounded by a phospholipid-unesterified cholesterol monolayer. Some aqueous spaces were seen within the particle core. Unesterified cholesterol-rich lipid particles were multilamellated, solid structures and vesicles comprised of single or multiple lamellas. The esterified cholesterol-rich particle had a density less than 1.01 g/ml, whereas the unesterified cholesterol-rich particle had a density between 1.03 and 1.05 g/ml. Both particles were similar in size (90% of both particles ranged in size between 40 to 200 nm in diameter) and had an unesterified cholesterol-to-phospholipid molar ratio of 2.5:1. The predominant phospholipid in both particles was sphingomyelin. The fatty acyl compositions of cholesteryl ester and phospholipid also were similar in both particles. Palmitate, oleate, and linoleate were the major fatty acids in the cholesteryl ester fraction, whereas palmitate, stearate, oleate, and linoleate were predominant in the phospholipid fraction. The origins and the role of these two unusual lipid particles in vessel wall cholesterol metabolism remain to be determined.     

Subset of Vascular Dendritic Cells Transforming into Foam Cells in Human Atherosclerotic Lesions

It has been previously demonstrated that S-100 positive vascular dendritic cells are involved in human atherosclerosis and they usually show a low level of accumulation of lipids in their cytoplasm, even though they located among foam cells and cellular debris in atherosclerotic lesions. During ongoing immunohistochemical investigations, however, we have found that a few S-100 positive cells exhibited a foam cell appearance. Therefore, we undertook an electronmicroscopic examination to see if any foam cells exhibit the distinctive features of vascular dendritic cells such as the presence of dense granules and a tubulovesicular system uniquely found in well differentiated dendritic cells. Foam cells exhibiting the typical characteristics of vascular dendritic cells were indeed found. Their cytoplasm contained a large number of lipid vacuoles and cisterns of the tubulovesicular system as well as dense granules which, in contrast to lysosomes present in macrophages, did not transform into phagolysosomes. The formation of a central lamina inside cisterns of the tubulovesicular system was also detected. These pentalaminal structures, comprised of two parallel limiting membranes and a central lamina, are similar to the Birbeck granules present in human epidermal Langerhans cells. From our present observations we speculate that the defense mechanisms against extensive lipid accumulation may be broken in some vascular dendritic cells, causing them to transform into foam cells.     

A Numerical Model to Study the Interaction of Vascular Stents with Human Atherosclerotic Lesions

A methodology is proposed that identifies optimal stent devices for specific clinical criteria. It enables to predict the effect of stent designs on the mechanical environment of stenotic arteries. In particular, we present a numerical study which is based on the interaction of a vascular stent with a patient-specific, atherosclerotic human iliac lesion of type V. The stress evolution in four different tissue components during and after stenting is investigated. The geometric model of the artery is obtained through MRI, while anisotropic material models are applied to describe the behavior of tissues at finite strains. In order to model the observed fissuring and dissection of the plaque under dilation, the undeformed configuration of the arterial wall incorporates two initial tears. The 3D balloon-stent-artery interaction problem is modeled by means of a contact algorithm, which is based on a C ²-continuous surface parametrization, hence avoiding numerical instabilities of standard facet-based techniques. In the simulations three different stent designs are studied. The performance of each stent is characterized by scalar quantities relating to stress changes in the artery, contact forces, and changes in lumen area after stenting. The study concludes by suggesting two optimal stent designs for two different clinically relevant parameters.     

Accumulation of Cholesteryl Ester in Atherosclerotic Lesions

This article reviews aspects of the molecular pathology of cholesteryl ester accumulation in atherosclerotic lesions. 1. Transcytosis of lipoproteins through a cultured endothelial monolayer. 2. Effects of platelets and PGI, on intercellular transport of endothelial cells. 3. Transformation of macrophages to foam cells. 4. Cholesteryl ester deposition in the extracellular space of atherosclerotic lesions. The development and use of novel monoclonal antlbodies recognizing atherosclerotic lesions and peroxidized lipoproteins prepared from then are also discussed. Acta Pathol Jpn 42: 625–631, 1992.     

Differential Effects of Pioglitazone on Advanced Atherosclerotic Lesions

This Correspondence relates to Atherosclerosis in LDLR-knockout Mice in Inhibited, but not Reversed, by the PPARy Ligand Pioglitazone (Am J Pathol 2009 174:2007–2014).     

Viral and Bacterial DNA in Carotid Atherosclerotic Lesions

Atherosclerosis is a major health problem in industrialised countries. Several studies have suggested an association exists between certain microorganisms and the development of atherosclerosis. The aim of the study presented here was to assess the presence of viral or bacterial DNA in carotid atherosclerotic lesions. Nucleic acids were extracted from 18 carotid atherosclerotic lesions that had been collected surgically. Polymerase chain reaction was used to screen for specific genomic DNA from Chlamydia pneumoniae, cytomegalovirus and herpes simplex virus types 1 and 2. An original approach, based on the amplification by PCR of conserved bacterial 16S rDNA nucleotide sequences was also used to detect any bacterial species. The amplification product was identified by sequencing. Chlamydia pneumoniae, cytomegalovirus and herpes simplex 2 DNA were not detected in any of the samples. Herpes simplex 1 DNA was detected in 3 of the 18 samples. Genes encoding bacterial 16S rRNA were amplified and sequenced in eight atherosclerotic lesions. DNA sequences were identified by comparison with sequences registered in the GenBank database. These eight carotid atherosclerotic lesions were shown to contain several bacterial species belonging to human flora or the environment. The exact role of these microorganisms in the genesis or development of the atherosclerotic lesions remains unclear, but they may increase the inflammatory process or be an epiphenomenon. Electronic Publication     

Identification of Inflamed Atherosclerotic Lesions In Vivo Using PET-CT

Inflammation plays a major pathogenetic role in the development of atherosclerotic plaques and related thromboembolic events. The identification of vulnerable plaques is of the utmost importance, as this may allow the implementation of more effective preventive and therapeutic interventions. Fluorodeoxyglucose positron emission tomography (FDG-PET) has been shown to be useful for tracing inflammation within plaques. However, its relationship to immunohistochemical findings in different territories of the peripheral circulation was not completely elucidated. We aimed to determine whether plaque inflammation could be measured by PET in combination with computer tomography (CT) using FDG and what is the relationship between FDG uptake and immunohistochemical findings in the removed atherosclerotic lesions of the femoral and carotid arteries. The study included 31 patients, 21 patients with high-grade stenosis of the internal carotid artery (ICA) and 10 patients with occlusion of the common femoral artery (CFA), all of whom underwent endarterectomy. Before endarterectomy in all patients, FDG-PET/CT imaging was performed. FDG uptake was measured as the maximum blood—normalized standardized uptake value, known as the target to background ratio (TBR max). TBR max amounted to 1.72?±?0.8, and in patients with ICA, stenosis was not significantly different from patients with CFA occlusion. Immunohistochemical and morphometric analyses of the plaques obtained at endarterectomy showed that the density of T lymphocytes and macrophages (number of cells per square millimeter) was significantly higher in subjects with stenosis of the ICA than in subjects with occlusion of the femoral arteries: lymphocytes, 1.26?±?0.21 vs. 0.77?±?0.29; p?=?0.02 and macrophages, 1.01?±?0.18 vs. 0.69?±?0.23; p?=?0.003. In the whole group of patients, the density of inflammatory cells significantly correlated with FDG uptake represented by PET-TBR max: T lymphocytes, r?=?0.60; p?<?0.01 and macrophages, r?=?0.65; p?<?0.01. The results of our study show that FDG uptake is related to the accumulation of inflammatory cells in atherosclerotic lesions. This finding suggests that FDG uptake reflects the severity of atherosclerotic vessel wall inflammation, and in stenotic lesions, it could be an indicator of their vulnerability. However, data from large outcome studies is needed to estimate the usefulness of this technique in identifying the most dangerous atherosclerotic lesions and vulnerable patients.     

Human Osteogenic Sarcoma: Fine Structure of the Fibroblastic Type

The fine structure of representative regions of nine fibroblastic osteogenic sarcomas was studied. As judged by light microscopic criteria, the tumors represented both highly malignant (grades 3-4) and less malignant (grade 2) varieties. By electron microscopy, six basic cell types were found in the selected regions (fibroblastlike, histiocytelike, and myofibroblastlike cells, along with xanthoma cells, multinucleated giant cells, and undifferentiated cells). In addition, occasional osteoblastlike cells were encountered. Fibroblastlike cells in general, and especially in grade 2 tumors, showed a fine structure, enabling differentiation from osteoblastlike cells. Multinucleated giant cells were of two morphologic types, and the fine structure appeared to be related to the malignancy potential and differentiation of the tumors. Many muitinucleated giant ceils in grade 2 tumors had a ruffled border and appeared to be highly active in digestive events (especially phagocytosis of whole cells and portions of cells). Accumulation of variable amounts of lipid in droplet form was common in the various types of cells present in the tissues. The observations were discussed with particular emphasis on the interrelationships and functional roles of the cells.     

Human Osteogenic Sarcoma: Fine Structure of Hard Tissue Areas

The structure of hard tissue areas (with osteoid and calcified matrix) in 10 osteoblastic, chondroblastic, and fibroblastic osteogenic sarcomas was studied in the electron microscope. Neoplastic cells commonly associated with these areas and presumably actively involved in the production of hard tissue were osteo-blastlike cells types 1 and 3, chondroblastlike cells type 1, and fibroblastlike cells, as defined and characterized in previous studies. The cells differed from those in soft tissue areas of osteogenic sarcomas in but one respect: they usually showed presence of irregular extrusions at their surfaces. Other types of osteoblastlike and chondroblastlike cells occurred rarely or not at all. Two types of multinucleated giant cells were recognized in these areas, one showing a fine structure reminiscent of that in osteoclasts, the other probably being of a neoplastic nature and engaged in the production of the calcifying matrix. The evidence suggested that neoplastic osteoblastlike, chondroblastlike, and fibrolastlike cells as well as certain multincleated giant cells might all be involved in the mineralization process and/or the formation of osteoid in osteogenic sarcomas. Although phenotypically of highly variable appearance, all these different cells may thus functionally (and probably histogenetically) be closely related.

The mineralization process in the tumor tissue appeared to be a modification of what occurs in normal ossification, possibly with an alternative or complementary pathway involving the production of spherical bodies with layered contents.     

Human Osteogenic Sarcoma: Fine Structure of the Osteoblastic Type

The fine structure of representative regions of 13 osteoblastic osteogenic sarcomas was studied. These regions contained four morphologically distinguishable subtypes of osteoblastlike cells. In addition, fibroblastlike and chondroblastlike cells were present, along with multinucleated giant cells, leukocytes, macrophagelike cells, and small populations of histogenetically unclassifiable (but probably neoplastic) cells.

The morphologic evidence was compatible with the view that the variations in appearance among the subgroups of osteobl astlike cells reflected differences in maturation and differentiation of these cells. In at least one subgroup, the morphologic findings suggested that the ceils were capable of manufacturing a secretory product. The multinucleated giant cells occurring in genuine tumor areas appeared to be closely related to neoplastic osteoblasts.

The presence of chondroblastlike cells in the tissues illustrates that cells with a diverging differentiation can occur in an osteoblast-dominated cell population. This agrees with the view that the neoplastic cells originate from a mesenchymal stem cell with potential for multifaceted differentiation.     

Inelasticity of Human Carotid Atherosclerotic Plaque

Little mechanical test data exists regarding the inelastic behavior of atherosclerotic plaques. As a result finite element (FE) models of stenting procedures commonly use hyperelastic material models to describe the soft tissue response thus limiting the accuracy of the model to the expansion stage of stent implantation and leave them unable to predict the lumen gain. In this study, cyclic mechanical tests were performed to characterize the inelastic behavior of fresh human carotid atherosclerotic plaque tissue due to radial compressive loading. Plaques were classified clinically as either mixed (M), calcified (Ca), or echolucent (E). An approximately linear increase in the plastic deformation was observed with increases in the peak applied strain for all plaque types. While calcified plaques generally appeared stiffest, it was observed that the clinical classification of plaques had no significant effect on the magnitude of permanent deformation on unloading. The test data was characterized using a constitutive model that accounts for both permanent deformation and stress softening to describe the compressive plaque behavior on unloading. Material constants are reported for individual plaques as well as mean values for each plaque classification. This data can be considered as a first step in characterizing the inelastic behavior of atherosclerotic plaques and could be used in combination with future mechanical data to improve the predictive capabilities of FE models of angioplasty and stenting procedures particularly in relation to lumen gain.     

The Fine Structure of Freeze-Fractured Blood Platelets

The present study has examined the fine structure of freeze-fractured human platelets. Advances in methods used for cell preparation and freezing, together with better instrumentation, have resulted in improved preservation of platelet ultrastructure. Aspects of surface membrane topography and the number, depth of penetration, tortuosity and fenestration of channels belonging to the open canalicular system were revealed in a manner which cannot be fully appreciated in sections of platelets. Elements of the dense tubular system of channels and masses of glycogen were identified in the platelet replicas. The technic may provide an important approach for defining the structural physiology and pathology of blood platelets.